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研究生:林隆誌
研究生(外文):Lung-JR Lin
論文名稱:溶菌蛋白質成熟過程對於大腸桿菌素分泌作用之影響
論文名稱(外文):Role of maturation of lysis protein in the processes of colicin release
指導教授:翟建富翟建富引用關係
指導教授(外文):Kin-Fu Chak
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:118
中文關鍵詞:大腸桿菌素溶菌蛋白質內膜通透性外膜通透性螢光染劑
外文關鍵詞:colicinlysis proteininner membrane permeabilityouter membrane permeabilityfluorescence dyeflow cytometry
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大腸桿菌素E7 (colicin E7),為大腸桿菌分泌之核酸型大腸桿菌素。由於不具有任何已知細菌分泌機制的訊息胜肽(Signal peptide),所以其分泌系統仍然未知。溶菌蛋白質(Lysis protein)為大腸桿菌基因群的脂蛋白質,扮演著協助大腸桿菌素分泌的作用,但其詳細機制亦仍然未知。利用螢光染劑追蹤溶菌蛋白質對於內外膜通透性的影響,明顯地指出脂化作用為造成內外膜通透性增加的關鍵。並且顯示出溶菌蛋白質不須透過活化外膜磷脂酶A (outer membrane phospholipase A),就可以造成外膜通透性增加。外膜通透性變化的時間差異與外膜磷脂酶A突變株的研究,更發現外膜磷脂酶A僅作用於分泌的後期,進一步造成Quasilysis。另外,以免疫電顯(Immuno-Electronic Microscopy)標定結果更顯示,溶菌蛋白質表現後,快速地成熟完成,並運送至外膜。外膜的結構也因溶菌蛋白質,而導致不穩定的現象。外膜與內膜因溶菌蛋白質,在特定區域形成點狀貼附,而非如噬菌體的溶菌作用一般,造成外膜全面崩解。所以本研究發現溶菌蛋白質改變內外膜通透性,大腸桿菌素E7得以藉此弱化內外膜作用,釋放至環境中。
Colicin E7 was one of nuclease type colicins secreting by Escherichia coli. It was encoded on pColE7-K317 plasmid that there were also encoded the inhibitor of colicin E7 (immunity protein) and a small lipoprotein (lysis protein). The mechanism of secretion of colicin E7 was still unclear. The small lipoprotein, the lysis protein, played the most important role in secretion of colicin E7. The mature lysis protein was translocated to the inner leaf of outer membrane and activated outer membrane phospholipase A to cause the quasis-lysis. We detected the ratio of extracellular and intracellular colicins by ELISA. It was found that the secretion of colicin was earlier than the decline in culture turbidity. The TO/PI staining showed that the permeability of inner membrane was increased in short time after induction to expression of lysis proteins. Point mutated lysis proteins was found that lipomodification of lysis protein made the inner membrane permeable. Increasing of the permeability of outer membrane was later than inner membrane detected by the NPN assay. The phospholipase A defective strain was found that the mature lysis protein increased the permeability of outer membrane independently. We also pointed that the secretion of colicin was happened in the OMPLA defective strain. The TO/PI staining and NPN assay showed that the maturation of lysis proteins increased permeability of membranes same with the wild-type strain. We found lower permeability of outer membrane in the OMPLA defective stain than the wide-type strain after long term induction. It indicated that OMPLA was major factor to make the quasislysis. The immuno-EM data showed that the lysis protein located on the inner leaf of outer membrane and polymerizated to make cell envelop fusion. Our observed that colicin E7 was secreted through fusion sites. The lysis protein disturbed host cell membrane and make colicin E7 secret to the environment.
中文摘要 I
ABSTRACT II
第一章 緒論 1
1.1 大腸桿菌素 1
1.2 溶菌蛋白質 3
1.2.1 溶菌蛋白質結構 4
1.2.2 蛋白質成熟機制 5
1.3 已知大腸桿菌素分泌機制 6
第二章 研究目的 8
第三章 材料方法 10
3.1 研究材料 10
3.1.1 菌株 10
3.1.2 培養基 10
3.1.3 質體 10
3.1.4 抗生素與誘導劑 10
3.1.5 抗體 11
3.1.6 緩衝液 11
3.2 研究方法 12
3.2.1 溶菌蛋白質表現載體建構與表現 12
3.2.2 點突變溶菌蛋白質基因建構 13
3.2.3 受容菌體製備與熱刺激轉形 13
3.2.4 菌落PCR篩選 13
3.2.5 大腸桿菌素E7分泌率測定 14
3.2.6 先驅脂蛋白質切割作用阻斷 15
3.2.7 內膜通透性分析 15
3.2.8 流式細胞儀分析 16
3.2.9 外膜通透性 17
3.2.10 西方墨點分析 17
3.2.11 免疫電顯 18
第四章 結果 20
4.1 菌液濁度下降非大腸桿菌素E7的分泌關鍵 20
4.2 溶菌蛋白質對於宿主細胞內外膜通透影響 21
4.3 脂化修飾造成內膜通透增加 24
4.4 外膜磷脂酶A造成菌液濁度下降 29
4.5 溶菌蛋白質獨自造成內外膜通透 31
4.6 溶菌蛋白質的s-diglycerylation即可對內膜造成擾動 35
4.7 溶菌蛋白質的次細胞位置變化 36
第五章 討論 38
5.1 大腸桿菌素E7非主要藉Quasilysis分泌至胞外 38
5.2 溶菌蛋白質直接促進大腸桿菌素E7分泌 39
5.3 溶菌蛋白質促進大腸桿菌素通過內膜 40
5.4 溶菌蛋白質的脂化作用造成內膜通透 41
5.5 溶菌蛋白質獨自造成外膜通透 43
5.6 溶菌蛋白質藉破壞內外膜通透進行分泌 45
第六章 結論 47
第七章 參考文獻 48
第八章 圖表結果 58
第九章 附錄 107
附錄A. 內膜通透變化標準螢光訊號分佈 108
附錄B. 溶菌蛋白質造成菌體顆粒性變化 110
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